Human therapeutic uses of BPI protein products

ABSTRACT

Disclosed are methods for treatment of humans exposed to bacterial endotoxin in circulation by administration of bactericidal/permeability-increasing (BPI) protein products. Serologically and hematologically verifiable alleviation of endotoxin mediated increases in circulating cytokines, fibrinolysis and coagulation factors and changes in lymphocyte counts are observed upon such treatment. Also observed is alleviation of endotoxin mediated decreases in systemic vascular resistance index (SVRI) and concomitant increases in cardiac index (CI).

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.08/291,112 filed Aug. 16, 1994, now U.S. Pat. No. 5,643,875 which is acontinuation-in-part of U.S. patent application Ser. No. 08/188,221,filed Jan. 24, 1994, abandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to therapeutic methods and moreparticularly to methods for treatment of humans exposed to bacterialendotoxin in blood circulation as a result of, e.g., Gram negativebacterial infection, accidental injection of endotoxin-contaminatedfluids, translocation of endotoxin from the gut, release of endotoxininto circulation as a result of antibiotic mediated bacterial cytolysisand the like.

Gram negative bacterial endotoxin plays a central role in thepathogenesis of gram negative sepsis and septic shock, conditions whichremain leading causes of morbidity and death in critically ill patients.Endotoxin interacts with inflammatory cells, releasing endogenousmediators such as cytokines, hydrolases, peptides, prostaglandins andother compounds that contribute to the pathophysiology of septic shock.While principal causes of entry of endotoxin into circulation are Gramnegative bacteremia and translocation of bacteria and bacterial productsfrom the gut, endotoxin also may enter circulation as the result ofaccidental injection of contaminated fluids or through release ofendotoxin from bacteria lysed as a consequence of antibiotic therapy. Inthe recent past, therapeutic methods proposed for treatment of sepsisand septic shock have had as their focus attempts to bind endotoxin incirculation and inhibit direct and indirect release into circulation ofproinflammatory substances mediated by the presence of endotoxin.Anti-endotoxin antibodies, for example, have shown promise in inhibitionof endotoxin effects in humans.

A difficulty consistently encountered in developing therapeutic methodsand materials for treatment of endotoxemia in humans has been thegeneral unreliability of in vitro and even non-human in vivo testresults as an indicator of human therapeutic potential. Because theeffects of endotoxin in circulation are complex and involve direct andindirect responses by many cell types in the body, test results inattempts to intervene in endotoxin's effects on a particular cell typein vitro present an incomplete basis for assessment of in vivo effects.Animal studies are complicated by differences in the effect of bacterialendotoxin on different animal species and in different models with thesame species. While humans are exquisitely sensitive to endotoxin, theresponses of other animals vary significantly. For example, mice andrats are far more resistant to endotoxin on a weight basis than arerabbits and dogs; a life-threatening dose in rabbits would produceminimal effects in mice. Moreover, the types of effects noted are quitevariable. Dogs display intestinal hemorrhages following sublethal butshock-producing doses of endotoxin while other commonly used laboratoryanimals do not. Mice housed at usual room temperature become hypothermicafter injection of endotoxin but develop fever when housed at 30° C.See, e.g., page xx in the Introduction in Cellular Biology of Endotoxin,L. Berry ed., Volume 3 in the series Handbook of Endotoxin (R. Proctor,series ed.) Elsevier, Amsterdam, 1985.

Of interest to the background of the invention are numerous reportsconcerning the in vivo effects of administration of endotoxin to healthyhuman volunteers. Martich et al., Immunobiol., 187:403-416 (1993)provides a current and detailed review of the literature addressing theeffects on circulatory system constituents brought about by experimentalendotoxemia in otherwise healthy humans. Noting that the responsesinitiated by endotoxin in humans are common to the acute inflammatoryresponse that is part of the host reaction to tissue injury orinfection, the authors maintain that administration of endotoxin servesas a unique means of evaluating inflammatory responses as well asresponses specific to endotoxin. The authors also note that, whileadministration of endotoxin to healthy humans is not a precise model forthe entirety of host responses in septic shock, it does allowinvestigation of the initial host inflammatory response to bacterialendotoxin.

Martich et al. note that intravenous administration of endotoxin isuniformly accompanied by a febrile response and various constitutionalchanges (myalgia and the like) which are attenuated by ibuprofen but notby the phosphodiesterase inhibitor, pentoxifylline. Cardiovascularresponses qualitatively similar to those observed in clinical sepsis areobserved in experimental endotoxemia in humans. Characteristic increasesare observed in circulating cytokines such as tumor necrosis factor α(TNF), interleukin 6 (IL-6); interleukin 1β (IL-1β), interleukin 8(IL-8), and granulocyte colony stimulating factor (GCSF). Inhibitorysoluble receptors of TNF were also noted to rise in a characteristicpattern following increases in levels of circulating TNF. The studiesreported on in Martich et al. provided observations that ibuprofenincreased levels of circulating TNF and IL-6 in experimental endotoxemiaand that pentoxifylline decreased circulating TNF, but not circulatingIL-6.

Human experimental endotoxemia was noted to give rise to humoralinflammatory responses similar to those observed in sepsis. Thefibrinolytic system is activated and levels of tissue plasminogenactivator (tPA) in circulation rise, accompanied by increases inα2-plasmin inhibitor-plasmin complexes (PAP), confirming activation ofplasminogen by tPA. Endotoxin administration to humans has been observedto prompt transitory leukopenia followed by rapid leukocytosis.Neutrophil degranulation occurs with attendant release of elastase(measured as elastase/α1-antitrypsin (EAA) complexes) and lactoferrininto circulation.

Martich et al. conclude that endotoxin administration to humansrepresents an important model of acute inflammation which reproducesmany of the inflammatory events that occur during sepsis and septicshock and provides a unique means of studying host responses to animportant bacterial product.

Following publication of the Martich et al. review article, the sameresearch group reported on a study of experimental endotoxemia whereinan attempt was made to ascertain whether endotoxin administration intocirculation could give rise to increased cytokine levels in the lung asmeasured by broncheoalveolar lavage (BAL). Boujoukos et al., J. Appl.Physiol., 74(6):3027-3033 (1993). Even when ibuprofen wasco-administered to enhance endotoxin mediated levels of circulating TNFand IL-6 in humans, no increases in TNF, IL-6 or IL-8 levels wereobserved in BAL fluid, suggesting that cytokine responses to endotoxinin circulation were compartmentalized and did not directly involve lungtissue endothelia.

Studies of the cardiovascular disturbances in septic shock haveestablished that shock is usually characterized by a high cardiac index(CI) and a low systemic vascular resistance index (SVRI). Parker et al.,Crit. Care. Med., 15:923-929 (1987); Rackow et al., Circ. Shock,22:11-22 (1987); and Parker et al., Ann. Intern. Med., 100:483-490(1984).! Of additional interest to the background of the invention arestudies of experimental endotoxemia in humans which have demonstrateddepression of myocardial contractility and diastolic dysfunction.Suffredini et al., N. Eng. J. Med., 321:280-287 (1989).!

Bactericidal/Permeability-Increasing protein (BPI) is a protein isolatedfrom the granules of mammalian polymorphonuclear neutrophils (PMNs),which are blood cells essential in the defense against invadingmicroorganisms. Human BPI protein isolated from PMNs by acid extractioncombined with either ion exchange chromatography Elsbach, J. Biol.Chem., 254:11000 (1979)! or E. coli affinity chromatography Weiss, etal., Blood, 69:652 (1987)! has optimal bactericidal activity against abroad spectrum of gram-negative bacteria. The molecular weight of humanBPI is approximately 55,000 daltons (55 kD). The amino acid sequence ofthe entire human BPI protein, as well as the DNA encoding the protein,have been elucidated in FIG. 1 of Gray et al., J. Biol. Chem., 264:9505(1989), incorporated herein by reference.

The bactericidal effect of BPI has been shown in the scientificliterature published to date to be highly specific to sensitivegram-negative species, with toxicity generally lacking for othermicroorganisms and for eukaryotic cells. The precise mechanism by whichBPI kills bacteria is not yet completely elucidated, but it is knownthat BPI must first attach to the surface of susceptible gram-negativebacteria. This initial binding of BPI to the bacteria involveselectrostatic and hydrophobic interactions between the basic BPI proteinand negatively charged sites on endotoxin. BPI binds to lipid A, themost toxic and most biologically active component of endotoxins.

In susceptible bacteria, BPI binding is thought to disruptlipopolysaccharide (LPS) structure, leading to activation of bacterialenzymes that degrade phospholipids and peptidoglycans, altering thepermeability of the cell's outer membrane, and initiating events thatultimately lead to cell death. Elsbach and Weiss, Inflammation: BasicPrinciples and Clinical Correlates, eds. Gallin et al., Chapter 30,Reven Press, Ltd. (1992)!. BPI is thought to act in two stages. Thefirst is a sublethal stage that is characterized by immediate growtharrest, permeabilization of the outer membrane and selective activationof bacterial enzymes that hydrolyze phospholipids and peptidoglycan.Bacteria at this stage can be rescued by growth in serum albuminsupplemented media. The second stage, defined by growth inhibition thatcannot be reversed by serum albumin, occurs after prolonged exposure ofthe bacteria to BPI and is characterized by extensive physiologic andstructural changes, including penetration of the cytoplasmic membrane.

Permeabilization of the bacterial cell envelope to hydrophobic probessuch as actinomycin D is rapid and depends upon initial binding of BPIto endotoxin, leading to organizational changes which probably resultfrom binding to the anionic groups in the KDO region of endotoxin, whichnormally stabilize the outer membrane through binding of Mg⁺⁺ and Ca⁺⁺.Binding of BPI and subsequent bacterial killing depends, at least inpart, upon the endotoxin polysaccharide chain length, with long O chainbearing organisms being more resistant to BPI bactericidal effects thanshort, "rough" organisms Weiss et al., J. Clin. Invest., 65:619-628(1980)!. This first stage of BPI action is reversible upon dissociationof the BPI from its binding site. This process requires synthesis of newLPS and the presence of divalent cations Weiss et al., J. Immunol., 132:3109-3115 (1984)!. Loss of bacterial viability, however, is not reversedby processes which restore the outer membrane integrity, suggesting thatthe bactericidal action is mediated by additional lesions induced in thetarget organism and which may be situated at the cytoplasmic membraneMannion et al., J. Clin. Invest., 86: 631-641 (1990)!. Specificinvestigation of this possibility has shown that on a molar basis BPI isat least as inhibitory of cytoplasmic membrane vesicle function aspolymyxin B In't Veld et al., Infection and Immunity, 56: 1203-1208(1988)! but the exact mechanism has not yet been elucidated.

A proteolytic fragment corresponding to the N-terminal portion of humanBPI holoprotein possesses essentialy all the bactericidal efficacy ofthe naturally-derived 55 kD human holoprotein. Ooi et al., J. Bio.Chem., 262: 14891-14894 (1987)!. In contrast to the N-terminal portion,the C-terminal region of the isolated human BPI protein displays onlyslightly detectable anti-bacterial activity. Ooi et al., J. Exp. Med.,174:649 (1991).! A BPI N-terminal fragment, comprising approximately thefirst 199 amino acid residues of the human BPI holoprotein and referredto as "rBPI₂₃," has been produced by recombinant means as a 23 kDprotein. Gazzano-Santoro et al., Infect. Immun., 60:4754-4761 (1992).!

Of additional interest to the present application are the disclosures ofreferences which relate to the potentiation of BPI bactericidal activityby 15 kD proteins derived from the granules of rabbit PMNs designatedp15. Ooi et al., J. Biol. Chem., 265:15956 (1990), disclose two related15 kD proteins derived from rabbit PMN granules which potentiate thefirst sublethal stage of BPI antibacterial activity but have aninhibitory effect on the second lethal stage of BPI antibacterialactivity. Levy et al., J. Biol. Chem., 268: 6058-6063 (1993), disclosethe sequences of cDNAs encoding the two rabbit proteins and report thatthe protein with the stronger potentiating effect reduces the requireddose of BPI for the early bacteriostatic effect by about 20-fold.

Of particular interest to the background of the present invention arereports of interaction between bacterial endotoxin and BPI proteinproducts in various in vitro and non-human in vivo assay systems. As oneexample, Leach et al., Keystone Symposia "Recognition of Endotoxin inBiologic Systems", Lake Tahoe, Calif., Mar. 1-7, 1992 (Abstract)reported that rBPI₂₃ (as described in Gazzano-Santoro et al., supra)prevented lethal endotoxemia in actinomycin D-sensitized CD-1 micechallenged with E. coli 011:B4 LPS. In additional studies Kohn et a., J.Infectious Diseases, 168: 1307-1310 (1993) demonstrated that rBPI₂₃ notonly protected actinomycin-D sensitized mice in a dose-dependent mannerfrom the lethal effects of LPS challenge but also attenuated theLPS-induced elevation of TNF and IL-1 in serum. Ammons et al.,Circulatory Shock, 41: 176-184 (1993)! demonstrated in a rat endotoxemiamodel that rBPI₂₃ produced a dose-dependent inhibition of hemodynamicchanges associated with endotoxemia. Kelly et al., Surgery, 114: 140-146(1993) showed that rBPI₂₃ conferred significantly greater protectionfrom death than an antiendotoxin monoclonal antibody (E5) in miceinnoculated intratracheally with a lethal dose of E. coli. Kung, et al.,International Conference on Endotoxin Amsterdam IV, Aug. 17-20 (1993)(Abstract) disclosed the efficacy of rBPI₂₃ in several animal modelsincluding live bacterial challenge and endotoxemia models.

M. N. Marra and R. W. Scott and co-workers have addressed endotoxininteractions with BPI protein products in U.S. Pat. Nos. 5,089,274 and5,171,739, in published PCT Application WO 92/03535 and in Marra et al.,J. Immunol., 144:662-665 (1990) and Marra et al., J. Immunol.,148:532-537 (1992). In vitro and non-human in vivo experimentalprocedures reported in these documents include positive assessments ofthe ability of BPI-containing granulocyte extracts, highly purifiedgranulocytic BPI and recombinant BPI to inhibit endotoxin stimulation ofcultures of human adherent mononuclear cells to produce tumor necrosisfactor α (TNF) when endotoxin is pre-incubated with the BPI product.Pre-incubation of endotoxin with BPI protein products was also shown todiminish the capacity of endotoxin to stimulate (upregulate) neutrophilcell surface expression of receptors for the complement systemcomponents C3b and C3bi in vitro. However, neither of these complementsystem components is known to have been demonstrated to be present inincreased amounts in circulation as a result of the presence ofendotoxin in human circulation. The experimental studies reported inthese documents included in vivo assessments of endotoxin interactionwith BPI protein products in test subject mice and rats. In one seriesof experiments, BPI was noted to inhibit stimulation of lung cellproduction of TNF (measured on the basis of cytotoxicity to fibrosarcomacells of broncheoalveolar lavage fluids) in mice challenged byintranasal administration of endotoxin. In another series ofexperiments, administration of BPI was noted to protect mice and ratsfrom lethal challenge with various bacterial endotoxin preparations andlive Pseudomonas and binding of BPI to endotoxin was noted to diminishpyrogenicity in rabbits. As noted above, however, Boujoukas et al., J.Appl. Physiol., 74(6):3027-3033 (1993) have demonstrated that, whileadministration of endotoxin to human circulation resulted in increasedlevels of circulating TNF, IL-6 and IL-8, no increases in thesesubstances could be detected in broncheoalveolar lavage fluids of thehuman subjects.

Since the filing of parent U.S. patent application Ser. No. 08/188,221on Jan. 24, 1994, additional studies of in vitro and in vivo effects ofBPI have been published. Fisher et al., Critical Care Med.,22(4):553-558 (1994) addressed studies in mice, rats and rabbits andconcluded that, "The exciting possibility thatbactericidal/permeability-increasing protein may be a specifictherapeutic agent to enhance the natural negative feedback mechanismsfor regulating endotoxin in humans is worth investigation." Marra etal., Critical Care Med., 22(4):559-565 (1994) addressed studies in miceand concluded that, "The potent endotoxin-binding and -neutralizingproperties of bactericidal/permeability-increasing protein indicate thatit might be useful in the treatment of endotoxin-related disorders inhumans."

Thus, while BPI protein products have been established to havepotentially beneficial interactions with endotoxin in a variety of invitro and non-human in vivo model systems, nothing is known concerningeffects of these products in humans actually exposed to bacterialendotoxin in circulation as a result of, e.g., Gram negative bacterialinfection, treatment with antibiotics, accidental injection withendotoxin-contaminated fluids, translocation of endotoxin from the gut,and the like.

SUMMARY OF THE INVENTION

The present invention provides novel methods for treatment of humansexposed to bacterial endotoxin in circulation involving theadministration of BPI protein products to provide serologically,hematologically and hemodynamically verifiable alleviation of endotoxineffects. The invention thus addresses the use of BPI protein products inthe manufacture of pharmaceutical compositions for the treatment ofhumans exposed to bacterial endotoxin in circulation.

According to one aspect of the invention, BPI protein products such asrBPI₂₃ are administered to humans in amounts sufficient to provideserologically, hematologically and hemodynamically verifiablealleviation of endotoxin mediated effects including, but not limited to:increases in circulating tumor necrosis factor (TNF), soluble TNFreceptors p55 and p75 sTNFr (p55) and sTNFr (p75)!, interleukin 6(IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10) and increasedneutrophil degranulation characterized by increased circulatinglactoferrin and/or elastase/α1 antitrypsin complexes (EAA); increases incirculating tissue plasminogen activator antigen (tPA Ag), tissueplasminogen activator activity (tPA act), and α2-plasmininhibitor-plasmin (PAP) complexes, plasminogen activator inhibitorantigen (PAI Ag) and urokinase type plasminogen activator (uPA);decreases in lymphocytes; increases in thrombin/antithrombin III (TAT)complexes; and decreases in systemic vascular resistance index (SVRI)and increases in cardiac index (CI).

According to another aspect of the invention BPI protein products areconjointly administered to human patients receiving antibiotic therapyto ameliorate, in a serologically, hematologicaly and hemodynamicallyverifiable manner, the effects of endotoxin release into circulationnormally attending antibiotic mediated cytolysis or breakdown ofbacteria.

In its presently preferred form, BPI protein products are administeredaccording to the invention in dosage amounts of about 0.1 to about 10mg/kg of body weight by parenteral, e.g., intravenous, routes in singleand multiple dosage formats or by continuous infusion. Oral andaerosolized administration is also within the contemplation of theinvention.

The present invention provides a use of a BPI protein product for themanufacture of a medicament for treatment of humans exposed to bacterialendotoxin in circulation. This aspect of the invention contemplates useof a BPI protein product in the manufacture of such medicaments in anamount effective to alleviate endotoxin mediated increase in circulatingtumor necrosis factor and interleukin 6; in an amount effective toalleviate endotoxin mediated increase in circulating interleukin 8 andin neutrophil degranulation as characterized by increased circulatinglactoferrin and/or elastase/α1 antitrypsin complexes; in an amounteffective to alleviate endotoxin mediated changes in numbers ofcirculating lymphocytes; in an amount effective to alleviate endotoxinmediated increase in circulating tissue plasminogen activator and tissueplasminogen activator activity; and in an amount effective to alleviateendotoxin-mediated decreases in systemic vascular resistance index. Thisaspect of the invention further contemplates use of a BPI proteinproduct in combination with bacterial antibiotics in the manufacture ofsuch medicaments.

Other aspects and advantages of the present human treatment methodinventions will be apparent to those skilled in the art uponconsideration of the following detailed description of presentlypreferred embodiments thereof, reference being made to the drawingwherein data is presented for human patients exposed to bacterialendotoxin and either placebo-treated (open circles) or treated with aBPI protein product according to the invention (filled circles). InFIGS. 2-19, the data points for the placebo-treated patients (opencircles) are offset on the time axis slightly to the right of thecorresponding data points for the BPI-treated patients (filled circles)for ease of visual comparison.

FIG. 1 is a graphic representation of the results of serologicalanalysis for bacterial endotoxin;

FIG. 2 is a graphic representation of the results of serologicalanalysis for TNF;

FIG. 3 is a graphic representation of the results of serologicalanalysis for soluble TNF p55 receptor;

FIG. 4 is a graphic representation of the results of serologicalanalysis for soluble TNF p75 receptor;

FIG. 5 is a graphic representation of the results of serologicalanalysis for IL-6;

FIG. 6 is a graphic representation of the results of serologicalanalysis for IL-8;

FIG. 7 is a graphic representation of the results of serologicalanalysis for IL-10;

FIG. 8 is a graphic representation of the results of serologicalanalysis for lactoferrin;

FIG. 9 is a graphic representation of the results of serologicalanalysis for elastase/α1 antitrypsin complexes;

FIG. 10 is a graphic representation of the results of hematologicalanalysis for neutrophils;

FIG. 11 is a graphic representation of the results of hematologicalanalysis for lymphocytes;

FIG. 12 is a graphic representation of the results of serologicalanalysis for tissue plasminogen activator antigen;

FIG. 13 is a graphic representation of the results of serologicalanalysis for tissue plasminogen activator activity;

FIG. 14 is a graphic representation of the results of serologicalanalysis for plasminogen activator inhibitor antigen;

FIG. 15 is a graphic representation of the results of serologicalanalysis for α2-plasmin inhibitor-plasmin complexes;

FIG. 16 is a graphic representation of the results of serologicalanalysis for urokinase type plasminogen activator;

FIG. 17 is a graphic representation of the results of serologicalanalysis for thrombin/antithrombin III (TAT) complexes.

FIG. 18 is a graphic representation of the results of analysis ofsystematic vascular resistance index (SVRI); and

FIG. 19 is a graphic representation of the results of analysis ofcardiac index (CI).

DETAILED DESCRIPTION

As used herein, "BPI protein product" includes naturally andrecombinantly produced BPI protein; natural, synthetic, and recombinantbiologically active polypeptide fragments of BPI protein; biologicallyactive polypeptide variants of BPI protein or fragments thereof,including hybrid fusion proteins and dimers; and biologically activepolypeptide analogs of BPI protein or fragments or variants thereof,including cysteine-substituted analogs. The BPI protein productsadministered according to this invention may be generated and/orisolated by any means known in the art. U.S. Pat. No. 5,198,541, thedisclosure of which is hereby incorporated by reference, disclosesrecombinant genes encoding and methods for expression of BPI proteinsincluding recombinant BPI holoprotein, referred to as rBPI₅₀ andrecombinant fragments of BPI. Co-owned, copending U.S. patentapplication Ser. No. 07/885,501 and a continuation-in-part thereof, U.S.patent application Ser. No. 08/072,063 filed May 19, 1993 which arehereby incorporated by reference, disclose novel methods for thepurification of recombinant BPI protein products expressed in andsecreted from genetically transformed mammalian host cells in cultureand discloses how one may produce large quantities of recombinant BPIproducts suitable for incorporation into stable, homogeneouspharmaceutical preparations.

Biologically active fragments of BPI (BPI fragments) includebiologically active molecules that have the same amino acid sequence asa natural human BPI holoprotein, except that the fragment molecule lacksamino-terminal amino acids, internal amino acids, and/orcarboxy-terminal amino acids of the holoprotein. Nonlimiting examples ofsuch fragments include an N-terminal fragment of natural human BPI ofapproximately 25 kD, described in Ooi et al., J. Exp. Med., 174:649(1991), and the recombinant expression product of DNA encodingN-terminal amino acids from 1 to about 193 or 199 of natural human BPI,described in Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992),and referred to as rBPI₂₃. In that publication, an expression vector wasused as a source of DNA encoding a recombinant expression product(rBPI₂₃) having the 31-residue signal sequence and the first 199 aminoacids of the N-terminus of the mature human BPI, as set out in FIG. 1 ofGray et al., supra, except that valine at position 151 is specified byGTG rather than GTC and residue 185 is glutamic acid (specified by GAG)rather than lysine (specified by AAG). Recombinant holoprotein (rBPI)has also been produced having the sequence (SEQ. ID NOS: 1 and 2) setout in FIG. 1 of Gray et al., supra, with the exceptions noted forrBPI₂₃ and with the exception that residue 417 is alanine (specified byGCT) rather than valine (specified by GTI). Other examples includedimeric forms of BPI fragments, as described in co-owned and co-pendingU.S. patent application Ser. No. 08/212,132, filed Mar. 11, 1994, thedisclosure of which is hereby incorporated by reference.

Biologically active variants of BPI (BPI variants) include but are notlimited to recombinant hybrid fusion proteins, comprising BPIholoprotein or a biologically active fragment thereof and at least aportion of at least one other polypeptide, and dimeric forms of BPIvariants. Examples of such hybrid fusion proteins and dimeric forms aredescribed by Theofan et al. in co-owned, copending U.S. patentapplication Ser. No. 07/885,911, and a continuation-in-part applicationthereof U.S. patent application Ser. No. 08/064,693 filed May 19, 1993which are incorporated herein by reference in their entirety and includehybrid fusion proteins comprising, at the amino-terminal end, a BPIprotein or a biologically active fragment thereof and, at thecarboxy-terminal end, at least one constant domain of an immunoglobulinheavy chain or allelic variant thereof.

Biologically active analogs of BPI (BPI analogs) include but are notlimited to BPI protein products wherein one or more amino acid residuehas been replaced by a different amino acid. For example, co-owned,copending U.S. patent application Ser. No. 08/013,801 (Theofan et al.,"Stable Bactericidal/Permeability-Increasing Protein Products andPharmaceutical Compositions Containing the Same," filed Feb. 2, 1993),the disclosure of which is incorporated herein by reference, disclosespolypeptide analogs of BPI and BPI fragments wherein a cysteine residueis replaced by a different amino acid. A preferred BPI protein productdescribed by this application is the expression product of DNA encodingfrom amino acid 1 to approximately 193 or 199 of the N-terminal aminoacids of BPI holoprotein, but wherein the cysteine at residue number 132is substituted with alanine and is designated rBPI₂₁ .increment.cys orrBPI₂₁.

Other BPI protein products useful according to the methods of theinvention are peptides derived from or based on BPI produced byrecombinant or synthetic means (BPI-derived peptides), such as thosedescribed in co-owned and copending U.S. patent application Ser. No.08/209,762, filed Mar. 11, 1994, which is a continuation-in-part of U.S.patent application Ser. No. 08/183,222, filed Jan. 14, 1994, which is acontinuation-in-part of U.S. patent application Ser. No. 08/093,202,filed Jul. 15, 1993), which is a continuation-in-part of U.S. patentapplication Ser. No. 08/030,644, filed Mar. 12, 1993, the disclosures ofwhich are hereby incorporated by reference. Other useful BPI proteinproducts include peptides based on or derived from BPI which aredescribed in co-owned and co-pending U.S. patent application Ser. No.08/274,299, filed Jul. 11, 1994, by Horwitz et al. and U.S. patentapplication Ser. No. 08/273,540, filed Jul. 11, 1994, by Little et al.

Presently preferred BPI protein products include recombinantly-producedN-terminal fragments of BPI, especially those having a molecular weightof approximately between 21 to 25 kD such as rBPI₂₃ or rBPI₂₁ anddimeric forms of these N-terminal fragments. Additionally, preferred BPIprotein products include rBPI₅₀ and BPI-derived peptides.

It is also contemplated that the BPI protein product be administeredwith other products that potentiate the activity of BPI proteinproducts. For example, serum complement potentiates the gram-negativebactericidal activity of BPI protein products; the combination of BPIprotein product and serum complement provides synergisticbactericidal/growth inhibitory effects. See, e.g., Ooi et al. J. Biol.Chem., 265: 15956 (1990) and Levy et al. J. Biol. Chem., 268: 6038-6083(1993) which address naturally-occurring 15 kD proteins potentiating BPIantibacterial activity. See also co-owned, co-pending U.S. patentapplication Ser. No. 08/093,201, filed Jul. 14, 1993, andcontinuation-in-part, U.S. patent application Ser. No. 08/274,373, filedJul. 11, 1994 which describes methods for potentiating gram-negativebactericidal activity of BPI protein products by administeringlipopolysaccharide binding protein (LBP) and LBP protein products. Thedisclosures of these applications are incorporated by reference herein.LBP protein derivatives and derivative hybrids which lack CD-14immunostimulatory properties are described in co-owned, co-pending U.S.patent application Ser. No. 08/261,660, filed Jun. 17, 1994 as acontinuation-in-part of U.S. patent application Ser. No. 08/079,510,filed Jun. 17, 1993, the disclosures of which are incorporated byreference herein. See also, the disclosure of immunoassays for thequantification of LBP disclosed in co-owned and co-pending U.S. patentapplication Ser. No. 08/377,391, filed Jan. 24, 1995 as acontinuation-in-part of U.S. patent application Ser. No. 08/186,811filed Jan. 24, 1994, the disclosures of which are incorporated byreference herein.

Practice of the methods of the present invention is illustrated in thefollowing examples wherein: Example 1 presents the protocol for acontrolled, double-blind crossover study of the effects of a BPI proteinproduct on human volunteers challenged with bacterial endotoxin; Example2 addresses serological analysis for cytokines and cytokine-relatedsubstances; Example 3 addresses lactose and glucose serologicalanalysis; Example 4 addresses total and differential leukocyte analysisand leukocyte activation analysis; Example 5 addresses serologicalanalysis of various coagulation and fibrinolysis parameters; and Example6 addresses analysis of hemodynamic parameters, specifically leftventricular function.

EXAMPLE 1

A controlled, double-blind crossover study was designed to investigatethe effects of BPI protein products (represented by therecombinant-produced amino terminal fragment referred to as rBPI₂₃) inhumans rendered endotoxemic by intravenous infusion of bacterialendotoxin.

Eight healthy male volunteers participated in the study. Each had anunremarkable medical history, a normal physical exam and essentiallynormal results in routine laboratory tests. EKG, chest X-ray andechocardiogram results were within the normal range for all volunteers.Participation in the study was excluded if any infectious disease hadoccurred within one month prior to the study, if any acute illness wasreported one week prior to the study or if any medication was takenwithin two weeks prior to the study. Written informed consent wasobtained from all participants and the study was approved by an ethicalreview board. The study had a cross over design in which the subjectswere challenged with endotoxin on two occasions, separated by a 6 weekwash out period. Placebo or rBPI₂₃ was administered concurrently withendotoxin at either of the two occasions, so that each volunteer servedas his own control. The treatment sequence (rBPI₂₃ followed by placebo,placebo followed by rBPI₂₃) was determined by randomization in a 1:1ratio to the two sequences. Thus four volunteers were treated withrBPI₂₃ during the first cycle of endotoxin challenge, and four duringthe second cycle.

The study drug, rBPI₂₃, was prepared according to the method ofGazzano-Santoro et al., supra, and was supplied as a clear, colorless,sterile, non-pyrogenic solution in 10 ml single use glass vials at aconcentration of 1 mg/ml in a buffer of 20 mmol/l sodium citrate, 0.15Msodium chloride, 0.1% poloxamer 188, 0.002% polysorbate 80, pH 5.0,containing no preservative. The placebo solution was supplied as aclear, colorless, sterile, non-pyrogenic solution in 10 ml single useglass vials. This solution contained 0.2 mg/ml human albumin in a bufferof 20 mmol/l sodium citrate, 0.15 mol/l sodium chloride, pH 5.0,containing no preservative. A treatment kit for each subject was codedaccording to the randomization schedule.

The endotoxin preparation used was FDA lot EC-5 (Escherichla coli),kindly provided by Dr. D. Hochstein (Bureau of Biologics, U.S. Food andDrug Administration, Bethesda, Md.). Before injection, the endotoxinpreparation was reconstituted according to FDA directions, warmed to 37°C., vigorously shaken for 30 minutes, and diluted to the appropriateconcentration in endotoxin-free saline. rBPI₂₃ or placebo wereadministered in exactly five minutes, in a forearm vein. Endotoxin wasinjected intravenously in one minute, in a forearm vein of the oppositearm, at a dose of 40 EU/kg during the third minute of the test druginfusion. Blood pressure and heart rate were monitored at 15 minuteintervals using a dinamap device (Critikon, Tampa, Fla.).

Blood was obtained through an indwelling intravenous catheter, at thefollowing time points: -30, 3, 5, 7, 12, 20, 30, 35 minutes, 1, 1.5, 2,3, 4, 6, 8, 10 and 12 hours. Blood was collected in Vacutainer tubes(Becton Dickinson, Rutherford, N.J.) containing acid citrate dextrosefor the BPI determination, in pyrogen-free plastic tubes (Falcon 2063,Oxnard, Calif.) containing pyrogen-free heparin (Thromboliquine®Organon, Oss, Netherlands, final concentration 30 U/ml) for theendotoxin test, in tubes containing K₃ -EDTA for leukocyte counts, andin siliconized Vacutainer tubes (Becton Dickinson) to which soybeantrypsin inhibitor was added for the elastase/α₁ -antitrypsin complex,lactoferrin and C3a-desarg assays. Serum was prepared for all othertests by centrifugation of clotted blood for 20 minutes at 2000×g.

For each experimental assessment made, data were analyzed to determineboth mean and median values and the associated standard errors. Resultswere plotted as a function of the relevant time period. Statisticalsignificance of treatment difference was assessed for median valuesusing the Wilcoxon signed rank test Lehmann, Nonparametrics-StatisticalMethods Based on Ranks, (Holden-Day, Inc., San Francisco, Calif. 1975)!on the percent change in trapezoidal area under the curve (AUC) for BPIprotein product treatment relative to the AUC on placebo treatment foreach volunteer. Percent change in AUC for rBPI₂₃ treatment relative toplacebo was defined as 100×(AUC_(BPI) -AUC_(placebo))/AUC_(placebo)). Anegative percent change implies a reduction in AUC for rBPI₂₃ treatmentrelative to placebo treatment. Statistical significance was assessedseparately in each functional group of assays so that the type 1 errorrate for each group of parameters was maintained at α=0.05. The Hochbergmethod Hochberg, Biometrika, 75:800-802 (1988)!, an improved Bonferroniprocedure Miler, Simultaneous Statistical Inference (Springer-Verlag,New York, N.Y., second ed., 1981)! for multiple significance testing,was used to determine statistical significance within each group ofparameters. Tests for carryover and period effects were performedaccording to the nonparametric method described by Koch, Biometrics,28:577-584 (1972). Where period effects were observed by this method,treatment differences were further explored by performing the test fortreatment effect in the presence of period effects as described in Koch,supra.

Infusion of rBPI₂₃ concomitantly with endotoxin resulted in a transient(average of 15 minutes) flush in six of the eight volunteers, but causedno other signs. Following infusion of endotoxin, oral temperature rosesimilarly after placebo or BPI infusion: following infusion of endotoxinand placebo, from 36.0°±0.2° C. to 37.8°±0.1° C.; with rBPI₂₃ infusion,from 35.6°±0.2° C. to 37.5°±0.3° C. The mean arterial blood pressuredecreased in both study periods, and was not influenced by the BPIinfusion. No safety related EKG changes were noted in any of thevolunteers. Volunteers suffered from clinical symptoms such as headache,myalgia, chills and nausea in both treatment regimens. All volunteerswere completely recovered at 24 hours following the start of theinfusion, and renal, liver and hematological parameters were within thenormal range at this time point.

Vital sign parameters including systolic blood pressure (SBP), diastolicblood pressure (DPP), mean arterial pressure (MAP), pulse, respirationrate and temperature were assessed. Table 1, below, sets out the resultsof statistical analysis of vital signs and reveals that there was nostatistically significant difference in values for the rBPI₂₃ -treatedpatients relative to placebo patients.

                  TABLE 1                                                         ______________________________________                                        VITAL SIGNS                                                                                    Median                                                                 AUC    % change         Statistical                                 Parameter hours  in AUC     p-value.sup.a                                                                       significance.sup.b                          ______________________________________                                        DBP       0-10   -1%        .95   NS                                          MAP       0-10   +1%        .74   NS                                          Resp Rate 0-10   -6%        .64   NS                                          SBP       0-10   +3%        .55   NS                                          Temp      0-10   -4%        .1094 NS                                          Pulse     0-10   -21%       .0391 NS                                          ______________________________________                                         .sup.a p-value comparing rBPI.sub.23 vs. placebo AUC within each subject      (Wilcoxon signed rank test).                                                  .sup.b Statistical significance as determined by the Hochberg method (S =     significant, NS = nonsignificant).                                       

Endotoxin assays were performed as described in van Deventer et al.,Blood, 76(12):2520-2526 (1990). Results are graphically represented inFIG. 1 wherein mean values±standard error of the mean forplacebo-treated patients are represented by open circles and values forBPI treated patients are represented by filled circles. Over the periodof zero time to 20 minutes, endotoxin levels were significantly higherfollowing placebo treatment in comparison to rBPI₂₃ treatment (Medianpercent change in AUC=98%; p-value=0.0156).

EXAMPLE 2 Cytokine and Cytokine Related Proteins

Serum levels of TNF were determined by immunoradiometric assay IRMAMedgenix, Fleurus, Belgium). Briefly noted, polypropylene tubes werecoated with a combination of monoclonal antibodies to recombinant TNFthat recognize distinct epitopes of TNF. The tubes were incubatedovernight with a mixture of the sample to be tested and anti-TNFantibody labeled with ¹²⁵ I. After decantation, the bound fraction wascounted in a gamma-counter, and the level of TNF was expressed in pg/mlin relation to a standard binding curve for recombinant TNF.

The serum concentrations of IL-1β, IL-8 and IL-10 were performed by acommercial ELISA kit according to the instructions of the manufacturer(Medgenix, Fleurus, Belgium). Serum concentration of IL-6 was alsodetermined by ELISA according to manufacturer's instructions. CentralLaboratory of the Netherlands Red Cross Blood Transfusion Service (CLB)Amsterdam, Netherlands!.

Soluble TNF receptor p55 and p75 concentrations were measured byspecific enzyme linked immunological assays as described in Van der Pollet al., J. Infect. Dis., 168:955-960 (1993). Briefly, microtiter plates(Maxisorp, Nunc, Denmark) were coated overnight at room temperature withTNF-binding non-inhibitory monoclonal antibodies against TNFR-p55 (clonehtr-20) or TNFR-p75 (clone utr-4), kindly provided by Dr. H. Gallati,Hoffmann LaRoche Ltd., Basel, Switzerland. Subsequently, the coatedwells were washed and the remaining protein-binding capacity of thewells was saturated with 1% BSA in 200 mM Tris/HCl, pH 7.5, 0.02% kathon(Hoffmann LaRoche Ltd., Basel, Switzerland). After discarding thestorage buffer, samples diluted 1:5 in 0.1M Tween 20, pH 7.25,containing 10% fetal bovine serum, 0.1% phenol, 0.1% Tween 20, 0.02%kathon were added to the wells. Standard curves were constructed withrecombinant sTNFR-p55 or sTNFR-p75. Peroxidase-conjugated recombinanthuman TNF was added and the mixtures were incubated for two days at 40°C. After washing, ortho-phenyldiamine-dihydrochloride substrate wasadded and incubated for 15-20 min. The reaction was stopped with 1.5M H₂SO₄ and the absorbance was spectrophotometrically determined at 490 nm.

Assay results for TNF and the p55 and p75 soluble TNF receptors aregraphically presented in FIGS. 2, 3 and 4, respectively. As indicatedtherein, TNF levels increased following endotoxin/placebo infusion from0.50±0.38 pg/ml (mean±SEM) immediately prior to infusion to peak levelsof 261.88±60.73 pg/ml at one and one half hours and returned to3.50±0.91 pg/ml by ten hours. When endotoxin infusion was accompanied byrBPI₂₃, however, the TNF peak was blunted (41.13±14.36 pg/ml) anddelayed to three hours after infusion. The serum concentrations of bothtypes of TNF receptors were elevated after endotoxin/placebo challenge(TNFR-p55: from 1.21±0.07 ng/mL at time 0 to 3.79±0.29 ng/mL at 3#hr;TNFR-p75: from 3.28±0.24 ng/mL at time 0 to 12.72±1.43 ng/mL at 3 hours)but increased to a lesser extent following endotoxin infusion withrBPI₂₃ (TNFR-p55: from 1.12±0.07 ng/mL at time 0 to 2.45±0.16 ng/mL at 4hours; TNFR-p75: from 3.27±0.29 at time 0 to 7.45±0.71 ng/mL at 6 hours)and peak levels were temporally shifted.

Post-treatment IL-1 levels were below the assay detection limit duringthe entire experiment in both treatment periods.

As indicated in FIG. 5, in endotoxin/placebo treated volunteers IL-6levels increased from 0.23±0.23 pg/ml (mean±SEM) prior to infusion to188.78±79.99 pg/ml at three hours and returned to normal values at 10hours. After rBPI₂₃ treatment, IL-6 rose from 0.06±0.04 pg/ml prior toinfusion to reach peak levels of 45.13±23.28 pg/mi at 4 hours andsubsequently returned to normal at 10 hours.

As indicated in FIG. 6, IL-8 increased from undetectable levels prior toinfusion to 69.83±16.72 pg/ml (mean±SEM) at two hours inendotoxin/placebo treated patients and decreased again to 4.18±2.38pg/ml at 10 hours following endotoxin infusion. After rBPI₂₃ infusionthe IL-8 peak occurred later (14.20±10.63 pg/ml at four hours) and IL-8levels remained undetectable throughout the entire ten hour assessmentperiod in three out of eight volunteers. At 10 hours, no IL-8 wasdetectable in any volunteer receiving rBPI₂₃.

Serum levels of IL-10 increased in endotoxin challenged volunteers afterboth placebo and rBPI₂₃ treatment. As seen in FIG. 7, peak levels wereobserved between one and one-half to three hours after infusion. In theplacebo group, IL-10 rose from 10.35±4.19 pg/ml (mean±SEM) prior toinfusion to 38.94±10.66 pg/ml (at three hours) and declined to10.35±4.37 pg/ml (at ten hours). In the rBPI₂₃ treated group, IL-10 wasincreased to a lesser extent; i.e., from an initial level of 8.86±3.35pg/ml to 21.76±6.89 pg/ml at three hours.

Table 2, below, sets out the results of statistical analysis of testresults for cytokines and cytokine related proteins from time 0 throughten hours.

                  TABLE 2                                                         ______________________________________                                        CYTOKINES AND CYTOKINE RELATED PROTEINS                                                         Median                                                                 AUC    % change         Statistical                                Parameter  hours  in AUC     p-value.sup.a                                                                       significance.sup.b                         ______________________________________                                        IL-1       0-10    0%        .25   NS                                         IL-10      0-10   -38%       .0156 S                                          IL-6       0-10   -79%       .0078 S                                          IL-8       0-10   -97%       .0078 S                                          TNF        0-10   -86%       .0078 S                                          TNF r(p55) 0-10   -40%       .0078 S                                          TNF r(p75) 0-10   -48%       .0078 S                                          ______________________________________                                         .sup.a p-value comparing rBPI.sub.23 vs. placebo AUC within each subject      (Wilcoxon signed rank test).                                                  .sup.b Statistical significance as determined by the Hochberg method (S =     significant, NS = nonsignificant).                                       

The above results establish that treatment of experimental endotoxemiain humans with a BPI protein product resulted in a serologicallyverifiable and statistically significant modification in cytokineresponse to the presence of endotoxin in circulation. The severity andtemporal setting of TNF production mediated by endotoxin wasdramatically altered and was accompanied by corresponding decreases inlevels of circulating soluble TNF receptors and a temporal shifting ofpeak circulating receptor values. In a like manner, the presence of IL-6in circulation as a consequence of endotoxin in circulation wassignificantly modified. While one previous study indicated thatcirculating TNF levels in experimental endotoxemia in humans could bediminished by intervention with pentoxifylline Zabel et al., Lancet,2:1474-1477 (1989)!, and ibuprofen has been observed to increase TNF andIL-6 levels following endotoxin administration, no previous study ofthis type has identified an agent which is effective in reducing TNF andIL-6 responses to endotoxin in humans.

Reduction in TNF response to endotoxin through administration of rBPI₂₃was accompanied not only by reductions in circulating TNF receptors butalso reduction in circulating IL-10, a substance which has beencharacterized as an anti-inflammatory cytokine. The reduction andshifting in peak IL-8 levels attending is the subject of discussion inExample 4, infra.

EXAMPLE 3 Lactate and Glucose Analysis

Lactate and glucose analyses were performed using standard laboratoryprocedures. Briefly summarized, serum glucose rose similarly over timein both treatment groups, while in each group there was only a slightrise from baseline levels of lactate.

EXAMPLE 4 Total and Differential Leukocytes and Leukocyte ActivationAnalysis

Leukocyte total and differential counts were determined through use of aflow cytometer (Technicon H1 system, Technicon Instruments, Tarrytown,N.Y.). Leukocytes counts decreased from 5.7±0.7×10⁹ /L at time 0 to4.0±0.6×10⁹ /L at 1.5 hours in the placebo period, and subsequently roseto 10.6±0.9×10⁹ /L at 6 hours. rBPI₂₃ blunted both the early leukocytedecrease (leukocyte count at 1.5 hours: 5.9±0.6×10⁹ /L, median change inAUC in the first two hours=+30%, p=0.078), and the subsequent rise inwhite blood cells (8.1±0.8×10⁹ /L at 6 hours) so that the medianreduction in leukocyte AUC in the first 12 hours was 19% (p=0.039).Marked monocytopenia and lyumphocytopenia developed within the first twohours following infusion of endotoxin, and monocyte and lymphocytecounts returned to baseline levels at 6 and 12 hours, respectively. Inthe placebo period, monocyte and lymphocyte counts became depressedagain at 24 hours following endotoxin challenge (lymphocytes:1.3±0.2×10⁹ /L; monocytes 0.4±0.1×10⁹ /L). Eosinophil counts decreasedfollowing endotoxin infusion with placebo treatment from 0.23 ±0.07×10⁹/L at time 0 to 0.04±0.008×10⁹ /L at 8 hours. Neutrophil counts rose inplacebo treatment from 3.5±0.6×10⁹ /L at time 0 to a peak level of 9.5±0.8×10⁹ /L at 6 hours, and subsequently decreased to 2.4±0.3×10⁹ /L at24 hours. As shown in Table 3 below, rBPI₂₃ infusion significantlyblunted the decrease of lymphycytes (median % change in AUC: +34%;p=0.0078) and blunted the monocytes, eosinophils and neutrophils withmedian % change on AUC for monocytes of +42% p=0.023!, for eosinophils:+37% p=0.039! and for neutrophils -26% p =0.016!. At 24 hours, however,monocyte, lymphocyte and eosinophil counts remained at baseline levelsfollowing rBPI₂₃ treatment (lymphocytes: 1.5±0.2×10⁹ /L; monocytes:0.5±0.1×19⁹ I; eosinophils: 0.15 ±0.04×10⁹ /L; neutrophils: 3.2±0.7×10⁹/L).

Results for neutrophil and lymphocyte determinations (mean±SEM) are alsoset out in graphic form in FIGS. 10 and 11, respectively. Table 3,below, sets out the results of statistical analysis of total anddifferential leukocytes from time 0 through 12 hours.

                  TABLE 3                                                         ______________________________________                                        LEUKOCYTE DIFFERENTIAL                                                                           Median                                                                 AUC    % change         Statistical                               Parameter   hours  in AUC     p-value.sup.a                                                                       Significance.sup.b                        ______________________________________                                        Basophils   0-12    +2%       .95   NS                                        Eosinophils 0-12   +37%       .0391 NS                                        WBC         0-12   -19%       .0391 NS                                        Monocytes   0-12   +42%       .0234 NS                                        Neutrophils 0-12   -26%       .0156 NS                                        Lymphocytes 0-12   +34%       .0078 S                                         ______________________________________                                         .sup.a p-value comparing rBPI.sub.23 vs. placebo AUC within each subject      (Wilcoxon signed rank test).                                                  .sup.b Statistical significance as determined by the Hochberg method (S =     significant, NS = nonsignificant).                                       

Plasma concentrations of elastase/α1-antitrypsin complex (EAA) andlactoferrin were measured by radioimmunoassay according to the methoddescribed in Nuijens et al., J. Lab. Clin. Med., 119:159-168 (1992), andvalues determined in terms of ng/ml. Complement activation was assessedby measuring the plasma concentrations of C3a-desarg using aradioimmunoassay as described in Hack et al., J. Immunol. Meth.,107:77-84 (1988) and values were expressed in terms of nmol/l.

Lactoferrin and EAA analysis results are set out in Table 4 below andmean±SEM results also graphically represented in FIGS. 8 and 9,respectively. Elastase-α₁ -antitrypsin complexes (EAA) after placebotreatment increased from 62.5±8.5 ng/mL at time 0 to 141±20.9 ng/mL at 4hours after endotoxin infusion, representing a rise to 2.26 timesbaseline. In the rBPI₂₃ treatment, only a minor increase in elastase/α₁-antitrypsin complex formation was observed (57.5±7.4 ng/mL at time 0 to96.0±8.7 ng/mL at 6 hours after infusion, a rise to 1.67 timesbaseline). Lactoferrin concentrations increased to 2.59 times baselineafter endotoxin infusion (from 163±22.6 ng/mL at time 0 to 422±58.0ng/mL at 4 hours). After rBPI₂₃ treatment, lactoferrin concentrationsincreased to only 1.64 times baseline (from 169±27.6 ng/mL at time 0 to276.6±60.5 ng/mL at 3 hours). The 47% reduction in lactoferrin AUC onrBPI₂₃ relative to placebo was significant (p=0.0078) (Table 4). The 36%reduction in EAA AUC was not significant (p=0.15); however, since thetest for period effect was significant for this parameter, the treatmenteffect was further explored by the method described by Koch, supra. Thisanalysis suggested a significant treatment effect of rBPI₂₃ in loweringEAA AUC (p-0.0304) (Table 4).

                  TABLE 4                                                         ______________________________________                                        LEUKOCYTE ACTIVATION                                                                            Median                                                                 AUC    % change         Statistical                                Parameter  hours  in AUC     p-value.sup.a                                                                       significance.sup.b                         ______________________________________                                        EAA        0-10   -36%       .0304.sup.c                                                                         S                                          Lactoferrin                                                                              0-10   -47%       .0078 S                                          ______________________________________                                         .sup.a p-value comparing rBPI.sub.23 vs. placebo AUC within each subject      (Wilcoxon signed rank test).                                                  .sup.b Statistical significance as determined by the Hochherg method (S =     significant, NS = nonsignificant).                                            .sup.c Accounting for period effect.                                     

While no significant differences in C3a-desarg were noted for the twotreatment strategies, in BPI-treated volunteers EAA complex formationand lactoferrin assay results were markedly lower than inplacebo-treated volunteers. The results observed in the EAA andlactoferrin assays are consistent with the previously noted reduction inIL-8 values in BPI-treated individuals. IL-8 has been implicated ineffecting degranulation of neutrophils and rises in lactoferrin and EAAin circulation result from neutrophil degranulation. Thus, the presentstudy reflects the first known instances of intervention in experimentalendotoxemia in humans resulting in reduction of circulating levels ofendotoxin mediated IL-8 and a coordinated reduction of neutrophildegranulation as assessed by analysis of circulating lactoferrin andEAA.

EXAMPLE 5 Fibrinolysis and Coagulation Parameter Analysis

Serological analyses were performed to assess circulating levels ofD-dimer, prothrombin fragments F1+2 (Frag F1+2), plasminogen activatorinhibitor antigen (PAI Ag), plasminogen activator inhibitor activity(PA1 Act), α2-plasmin inhibitor-plasmin complexes (PAP), protein Cactivity (Prot. C Act), thrombin-antithrombin III (TAT) complex, a2-antiplasmin (AAP), plasminogen, tissue plasminogen activator antigen(tPA Ag), tissue plasminogen activator activity (tPA Act) and urokinasetype plasminogen activator (uPA). Blood was collected by separate venouspunctures from antecubital veins, before and at 1, 1.5, 2, 3, 4, 6 and10 hours after the start of the endotoxin infusion and through 12 hoursfor platelets. Blood for AAP, plasminogen, tPA Ag, uPA, PAI Ag, PAI Act,D-dimer, Prot. C Act, Frag F 1+2 and TAT complex measurements (9volumes) was collected in vacutainer tubes (Becton Dickinson,Rutherford, N.J.) containing 3.8% sodium citrate (1 volume). Blood fort-PA Act was collected in Biopool Stabilyte™ tubes (Biopool, Umea,Sweden) containing a low pH citrate anticoagulant which stabilizes t-PAactivity by blocking inhibition of t-PA of PAI. For the measurements ofPAP complexes, blood was collected in siliconized vacutainer tubes(Becton Dickinson, Plymouth, England) to which EDTA (10 mM, finalconcentration) and Soy Bean Trypsin Inhibitor (Sigma T-9003; finalconcentration: 0.1 mg/mL) was added to prevent in vitro complexformation. Tubes containing K₃ -EDTA were used to collect blood forplatelet counts. For all measurements except platelet counts, plasma wasprepared by centrifuging at 2000×g for 30 minutes at 15° C., after whichplasma was frozen at -70° C. until batchwise assessment was performed.

Platelet counts were determined with the use of a flow cytometer(Technicon H1 system, Technicon Instruments, Tarrytown, U.S.A.). Plasmalevels of TAT complexes and of Frag F 1+2 were measured with ELISA's(Behringwerke AG, Marburg, Germany) Teitel et al., Blood, 59: 1086-1096(1982)!. Results are expressed in ng/mL and nmol/L, respectively.Protein C activity was measured by an amidolytic assay, as described inSturk et al., Clin. Chim Acta, 165: 263-270 (1987). tPA Act was measuredby an amidolytic assay Verheijen et al., Thromb. Haemostasis, 48:266-269 (1982)!. Briefly, 25 μl of plasma was mixed to a final volume of250 μl with 0.1M TrisHCl, pH 7.5, 0.1% (v/v) Tween-80, 0.3 mM S-2251(Kabi Haematology, MoIndal, Sweden), 0.13M plasminogen and 0.12 mg/mlCNBr fragments of fibrinogen (Kabi Haematology, Molndal, Sweden). Theresults are expressed as IU/ml (first international standard of theWorld Health Organization).

PAI Act was measured with an amidolytic assay Chmielewska, et al.,Thromb. Res., 31: 427-436 (1983)! in which the samples were incubatedfor 10 min at room temperature with an excess of tissue-type plasminogenactivator. Part of the t-PA was inhibited by PAI, present in the sample,and formed inactive complexes. Residual t-PA activity was determined bysubsequent incubation, with 0.13 μM plasminogen (Kabi Haematology), 0.12mg/ml cyanogen bromide-digested fibrinogen fragments (t-PA stimulator,Kabi Haematology) and 0.1 mM S-2251 (Kabi Haematology). The amount ofplasmin generated in the incubation mixture, determined by theconversion of the chromogenic substrate, was inversely proportional tothe PAI Act in the sample. The results of the samples to be tested wererelated to the results of samples of PAI Act depleted plasma (KabiHaematology) to which fixed amounts of t-PA were added. Results wereexpressed in international units (IU), where 1 IU is the amount of PAIAct that inhibits 1 IU t-PA.

t-PA Ag and PAI Ag were assayed with ELISA's Holvoet et al., Thromb.Haemostasis, 54: 684 (1985)! (Asserachrom t-PA, Diagnostica Stago,Asnieres-sur-Seine, France and PAI-ELISA kit, monozyme, Charlottenlund,Denmark, respectively). Results are expressed in ng/ml. uPA was measuredwith a sandwich-ELISA Binnema et al., Thromb. Res., 43: 569 (1986)!. Theassay measures the urokinase-antigen present in plasma, irrespective ofits molecular form i.e. pro-urokinase, active urokinase and urokinase incomplex with inhibitors; the results are expressed in ng/ml. Plasminogenactivity and AAP were measured by automated amidolytic techniquesaccording to methods described in Peeters et al., Thromb. Res., 28: 773(1982). The results are expressed as percentages of normal. D-Dimer wasmeasured with an ELISA (Asserachrom D-Di, Diagnostica Stago,Asnieres-sur Seine, France) Elms et al., Thromb. Haemostasis, 50: 591(1983)!. Results are expressed in μg/ml. PAP complexes were measured bya RIA as described in Levi et al., J. Clin. Invest. 88: 1155-1160(1991). Briefly, specific mAbs, raised against inactivated and complexedα2-antiplasmin were coupled to sepharose beads and incubated with plasmasamples. After washing the sepharose with phosphate buffered saline,bound complexes were subsequently incubated with ¹²⁵ I labeled mAbs toplasmin. After another washing procedure, sepharose-bound radioactivitywas measured. As standards, serial dilution of plasma in which a maximalamount of PAP complexes was generated by incubation with two chainurokinase (Choay, Paris, France), after pre-incubation of the plasmawith methylamine to inactivate α2-macroglobulin were used. The resultsare expressed as nmol/L.

Statistical analysis (as described in Example 1) of the results of theseanalyses are reflected in Tables 5 and 6. The mean±SEM results of assaysof tissue plasminogen activator antigen (t-PA Ag), tissue plasminogenactivator activity (t-PA Act), tissue plasminogen activator inhibitorantigen (PAI Ag), α2-plasmin inhibitor-plasmin (PAP) complexes,urokinase type plasminogen activator (uPA) and thrombin/antithrombin III(TAT) complexes are graphically represented in FIGS. 12, 13, 14, 15, 16and 17, respectively.

                  TABLE 5                                                         ______________________________________                                        FIBRINOLYSIS                                                                                    Median                                                                 AUC    % change         Statistical                                Parameter  hours  in AUC     p-value.sup.a                                                                       significanc.sup.b                          ______________________________________                                        α2-antipl                                                                          0-10    -3%       .95   NS                                         d-dimer    0-10   -45%       .31   NS                                         plasminogen                                                                              0-10   -10%       .20   NS                                         PAI Activity                                                                             2-10   -51%       .0304.sup.c                                                                         NS                                         PAP Complex                                                                              0-10   -51%       .0078 S                                          uPA        0-10   -50%       .0078 S                                          PAI Ag     2-10   -52%       .0078 S                                          tPA Ag     1-10   -79%       .0078 S                                          tPA Activity                                                                             1-6    -57%       .0078 S                                          ______________________________________                                         .sup.a p-value comparing rBPI.sub.23 vs. placebo AUC within each subject      (Wilcoxon signed rank test).                                                  .sup.b Statistical significance as determined by the Hochberg method (S =     significant, NS = nonsignificant).                                            .sup.c Accounting for period effect.                                     

                  TABLE 6                                                         ______________________________________                                        COAGULATION                                                                                     Median                                                                 AUC    % change         Statistical                                Parameter  hours  in AUC     p-value.sup.a                                                                       significance.sup.b                         ______________________________________                                        Protein C Activity                                                                       0-10   -21%       .64   NS                                         F1 + 2     1-10   -31%       .0391 NS                                         TAT Complex                                                                              1-10   -36%       .0078 S                                          ______________________________________                                         .sup.a p-value comparing rBPI.sub.23 vs. placebo AUC within each subject      (Wilcoxon signed rank test).                                                  .sup.b Statistical significance as determined by the Hochberg method (S =     significant, NS = nonsignificant).                                       

In this study, endotoxin induced the activation of the coagulationsystem, consistent with Van Deventer et al., supra, and Levi et al., J.Clin. Invest., 93:114 (1994). Specifically, the infusion of endotoxinresulted in 7.3-fold and 7.4-fold increase in plasma levels ofTAT-complexes and prothrombin fragment F₁₊₂, respectively. Mean levelsof TAT complexes rose from 5.5±1.4 to 40.0±5.3 ng/mL, pleaking at 3hours after endotoxin administration and therafter gradually decreasing(FIG. 17). F₁₊₂ plasma levels reached their peaks at four hours afterinfusion with endotoxin (increasing from 0.78±0.10 nmol/L to 5.77±1.27nmol/L). No significant changes were seen in plasma levels of protein Cactivity.

Infusion with rBPI₂₃ resulted in a significant reduction inendotoxin-induced thrombin generation. Specifically, maximal F₁₊₂ levelswere 3.30±0.39 nmol/L after the administration of endotoxin incombination with rBPI₂₃ as compared to after the administration ofendotoxin alone; maximal TAT complex levels were 30.8±6.9 ng/mL aftgerthe combined endotoxin and rBPI₂₃ treatment (FIG. 17). Treatment withrBPI₂₃ had no effect on plasma levels of protein C activity (Table 6).The AUC for TAT complexes was significantly reduced on rBPI₂₃ (medianreduction 36%, p=0.0078) (Table 6). The AUC for F₁₊₂ was also reduced onrBPI₂₃ ; this effect did not reach significance according to theHochberg, supra, method (median reduction 31%, p=0.0391).

Platelet counts decreased in both treatment groups from a baseline levelof 206.5±15.0×10¹² on placebo to 181.6±13.2×10¹² at 4 hours and from190.4±16.9×10¹² to 174.8±10.7×10¹² at 6 hours following rBPI₂₃ treatment(resulting in a 7% reduction of AUC; p=0.19).

Additionally, in this study, endotoxin induced initial activiation ofthe fibrinolytic system followed by inhibition, in agreement withSuffredini et al., N. Engl. J. Med., 320:1165 (1989). Plasminogenactivating activity increased from 0.22±0.03 IU/mL to 2.14±0.13 IU/mL,reaching a peak at two hours after the administration of endotoxinalone. This increase in plasminogen activator activity (FIG. 13) wasparalleled by an increase in t-PA antigen (FIG. 12) and u-PA antigen(FIG. 16). Peak levels of t-PA antigen at u-PA antigen were 45.6±6.6ng/mL at 3 hours and 6.0±0.5 ng/mL at 2 hours, respectively. The rise ofplasminogen activity was followed by plasmin generation, as reflected byincreasing levels of PAP-complexes (FIG. 15) and of D-Dimer, whichincreased from 4.61±0.38 nmol/L to 12.4±2.1 mnol/mL at 2 hours and from217±117 ng/mL to 707±120 ng/mL at 10 hours, respectively. Plasma levelsof PAI activity and antigen (FIG. 14) remained unchanged up to two hoursafter endotoxin administration. Thereafter a rapid increase was seen upto 34.5±2.6 IU/mL and 225.8±1.3 ng/mL, respectively, both peaking at 4hours after the infusion. The increase in PAI was followed in aninstanteous decrease in t-PA activity and subsequent plasmin generation,as reflected by decreasing levels of PAP-complexes. Since the assays fort-PA and u-PA antigen also measure plasminogen activator in complex withits inhibitor (i.e. PAI), values of these parameters only graduallydecreased.

The simultaneous adminstration of rBPI₂₃ and endotoxin resulted in asubstantial reduction and delay of endotoxin-induced fibrinolyticactivity. Peak levels of plasminogen activator activity (0.61±0.2 IU/mL)and t-PA antigen (16.4±6.7 ng/mL) were reached at 3 and 4 hours,respectively. Upon administration of endotoxin in combination withrBPI₂₃, no increase in u-PA antigen was seen. The endotoxin-inducedrises of plasma levels of PAP-complexes and D-Dimer were also greatlyattenuated by the rBPI₂₃ infusion. Peak levels of PAP complexes andD-Dimer were 7.7±1.3 nmol/L at 3 hours and 531±119.5 ng/mL at 10 hours,respectively. The endotoxin-induced increase of PAI activity and antigenwas delayed and partly inhibited by the simultaneous administration ofrBPI₂₃. Peak levels of PAI activity and antigen were reached at 6 hoursafter infusion of endotoxin and rBPI₂₃. The combined administration ofendotoxin and rBPI₂₃ resulted in a maximal PAI activity level of22.6±2.9 IU/mL and a maximal PAI antigen level of 122.9±22.0 ng/mL. TheAUC for t-PA activity, t-PA antigen, uPA antigen, PAP complexes and PAIantigen were significantly reduced on rBPI₂₃ (p=0.0078 for each; medianAUC reductions varied from 50 to 79%; Table 5). The AUC for PAI activityand D-dimer were reduced on rBPI₂₃ treatment although not significantly(median reductions of 51%, p=0.0304 and 45%, p=0.31; Table 5). The AUCfor plasminogen and α2 antiplasmin were not changed by rBPI₂₃ treatmentTable 5).

The placebo/endotoxin results in FIGS. 12 through 17 confirm acoordinated serological response to endotoxin administration in thatshortly after tPA levels rise, there is sharp increase in tPA activitywhich precipitously drops upon increases in levels of circulatingplasminogen activator inhibitor. The results demonstrate coordinatedintervention in these related phenomena by treatment with a BPI-proteinproduct. Peak levels in tPA are diminished and temporally shifted and acorresponding drop in tPA activity and circulating plasminogen activatorinhibitor level is observed. Activation of plasminogen by tPA, asindicated by levels of circulating α2-plasmin inhibitor-plasmincomplexes (PAP), was diminished. Thus, the present study reflects thefirst instance of intervention in endotoxin mediated increase incirculating tPA and its activity in experimental endotoxemia in humans.

EXAMPLE 6 Protection From Endotoxin-Induced Hyperdynamic CirculatoryState

Circulatory state assessments were performed as follows: Echocardiogramswere performed using an Ultramark 9 echocardiography machine (AdvancedTechnology Laboratories (Bothell, Wash.)) with a 2.25 MHz phased arrayprobe that featured a steerable pulsed Doppler mode. All subjectsunderwent basal echocardiography studies at rest (basal) includingM-mode measurements, 2-D imaging from parasternal, apical and subcostalviews, color-coded Doppler imaging and pulsed Doppler measurements.Optimal parasternal and apical windows were obtained and marked on thesubject's skin. All gain settings, sample volume size and depth andDoppler output settings were noted for each subject and carefullyrepeated at each measurement. The first basal study was performedseveral weeks before the first day of the endotoxin infusion. On eachinfusion day a basal study was performed in the early morning one hourprior to the infusion. The fourth basal study was obtained six to eightweeks after the last infusion. Averages of these four echocardiogramscomprise the basal values. On the study days at timepoints 1:30, 2:30,4:00, 5:00, 8:00, 12:00 hours after start of infusion the followingmeasurements were performed: M-mode; Left Ventricular End DiastolicDiameter (LVEDD), Left Ventricular End Systolic Diameter (LVESD).Interventricular Septal Thickness (IVS), Left Posterior Wall Thickness(LVPW), Dimension of the Aortic Root (Ao) and the Left Atrium (LA),tracings of the mitral valve motion and the aortic valve motion. 2-D:Standard parasternal long-axis and short-axis views, apical, andsubcostal views were made to assess diameters, wall motion and aspect ofmitral, aortic and tricuspid valve apparatus. Careful measurement of thediameter of the left ventricular outflow tract (D_(lvot)) at itsnarrowest point approximately one centimeter below the aortic valve wasperformed in the parasternal long axis view on an early systolic stillframe after aortic valve opening. At every time point at least sixmeasurements were made. Color-Doppler: Mitral valve flow and tricuspidvalve flow and regurgitation were assessed if present. Pulsed Dopplermeasurement: At the basal studies and on the study days at time points0:30, 1:00, 1:30, 2:00, 2:30, 3:00, 3:30, 4:00, 4:30, 5:00, 6:00, 8:00,10:00, 12:00 hours after the start of infusion a pulsed Dopplermeasurement of the systolic flow in the center of the left ventricularoutflow tract from the apical window was obtained. Ten consecutiveDoppler flow tracings were recorded on videotape. Heart rate,temperature and blood pressure were simultaneously recorded.

Following completion of the study off-line, the Velocity Time Integral(VTI) or spectral area in meters given by the sum of Vi·₆₇ t were tracedfrom the videotapes. Also the maximal velocity V_(max) was noted fromthe VTI tracings. Averages were obtained from ten consecutive beats.

Equations employed to analyze results were as follows:

(1) Left Ventricular End Diastolic Volume (mL):

    LVEDV=(7.0/(2.4+LVEDD))×LVEDD.sup.3)/100;

(2) Left Ventricular End Systolic Volume (mL):

    LVESV=(7.0/(2.4+LVESD))×LVESD.sup.3)/100;

(3) Fractional Shortening (%):

    FS=(LVEDD-LVESD)/LVEDD)×100;

(4) Ejection Fraction (%):

    EF=(LVEDV-LVESV)/LVEDV)×100;

(5) Cross Sectional Area_(lvot) (cm²):

    CSA=π(D.sub.lvot).sup.2 /4;

(6) Stroke Volume (mL):

    SV=CSA×VTI×100;

(7) Cardiac Output (CO) (L):

    CO=SV×HR/1000

where HR is Heart Rate (beats per minute);

(8) Cardiac Index (L/min/m²):

    CI=CO/BSA

where BSA is Body Surface Area; and

(9) Body Surface Area (m²):

    BSA=(Height).sup.0.725 ×(Weight).sup.0.425 ×(0.007184)

(10) Systemic Vascular Resistance Index (dyne*sec/cm⁵ per square meter):

    SVRI=80*(Mean Arterial Pressure-6)/Cardiac Index.

Table 7, below, sets out the results of statistical analysis (asdescribed in Example 1) of the primary left ventricular functionparameters, SVRI and CI. Graphic representations of SVRI and CI data areprovided in FIGS. 18 and 19 respectively.

                  TABLE 7                                                         ______________________________________                                        PRIMARY LEFT VENTRICULAR FUNCTION                                                      Median                                                                        % change              Statistical                                    Parameter                                                                              in AUC.sup.a  p-value.sup.b                                                                         significance.sup.c                             ______________________________________                                        SVRI     +28%          .0304.sup.d                                                                           S                                              CI       -13%          .0156   S                                              ______________________________________                                         .sup.a AUC calculated from hours 0-6                                          .sup.b pvalue comparing rBPI.sub.23 vs. placebo within each subject           (Wilcoxon signed rank test).                                                  .sup.c Statistical significance as determined by the Hochberg method (S =     significant, NS = nonsignificant) applied to primary analysis parameters.     .sup.d Accounting for period effect.                                     

Table 8, below, sets forth data concerning percent change in medianresult values and p-values for the collateral (secondary) assessments ofother left ventricular function parameters.

                  TABLE 8                                                         ______________________________________                                        COLLATERAL LEFT VENTRICULAR FUNCTION                                                          Median                                                                        % change                                                      Parameter       in AUC.sup.a                                                                           p-value.sup.b                                        ______________________________________                                        AO               -4%     .55                                                  CO              -11%     .0156                                                EF              -10%     .64                                                  FS              -13%     .74                                                  HR              -27%     .0078                                                IVS              +3%     .55                                                  LA              -15%     .38                                                  LVEDD            -4%     .95                                                  LVEDV            -4%     .84                                                  LVESD           +13%     .31.sup.c                                            LVESV           +14%     .31.sup.c                                            LVPW             +4%     .84                                                  SV               +9%     .15                                                  VTI              +1%     .55                                                  ______________________________________                                         .sup.a AUC calculated from hours 0-6.                                         .sup.b pvalue comparing rBPI.sub.23 vs. placebo within each subject           (Wilcoxon signed rank test).                                                  .sup.c Accounting for periof effect.                                     

Heart rate showed an increase in both study periods but less in therBPI₂₃ treatment period. For the rBPI₂₃ treatment period, heart raterose from 56 ±2 beats/min at time 0 to a maximum of 77±5 at 4:30 hours,dropping to 73 ±3 at 6 hours, while for the placebo treatment periodheart rate rose more rapidly from 61±5 at time 0 to a maximum of 87±4 at3:30 hours and dropping to 75±3 by 6 hours (27% reduction in AUC fromtime 0 to 6 hours, p=0.0078). The difference initial heart rate betweenthe placebo and rBPI₂₃ period was mainly due to a difference in heartrate in one subject who started with heart rates of 87 and 74 at time 0and 0:30 hours, respectively during the first infusion day (placebo),probably because of anxiety, and who had a heart rate of 55 at time 0the second day.

Velocity time integrals (VT1) in the rBPI₂₃ treatment period showed anincrease at 0:30 hours and 1 hour possibly related to the inital flushthat six of the eight volunteers experienced. For the rBPI₂₃ treatmentperiod VTI was 0.210±0.009 m at time 0, 0.223±0.008 m at 0:30 hours and0.222±0.006 m at 0:30 hours. For the placebo treatment period, VTI was0.203±0.008 m at time 0, 0.207±0.006 m at 0:30 hours and 0.201±0.007 mat 1 hour. From 5 hours onwards, there were consistently lower heartrates and higher VTI's in the rBPI₂₃ period.

V_(max) showed an increase in both infusion periods but the increase wasattenuated in the rBPI₂₃ treatment period. (Median AUC 1.15 for rBPI₂₃,2.29 for Placebo, p=0.061 by Wilcoxom Rank Sum test). V_(max) wasexamined statistically only in period 1 due to the presence of carryovereffects between period 1 and 2. V_(max) rose from 1.05±0.076 m/s at time0 to 1.23±0.08 m/s at 3:30 hours in the rBPI₂₃ treatment period and from1.05±0.05 m/s at time 0 to 1.29±0.05 m/s at 3:30 hours in the placebotreatment period. The flow profile in the left ventricular outflow tractchanges showing higher velocities but shorter ejection times resultingin conserved VTI.

Cross sectional area (CSA) was measured six times at each timepoint andaveraged CSA did not change with heart rate and the average value of allmeasurements of the CSA of one infusion period for each subject was usedas a constant in the equations. Average CSA in this group of eightvolunteers was 4.37±0.4 CM².

Cardiac index increased in both study periods but rBPI₂₃ infusiondiminshed the endotoxin-induced rise in cardiac index significantly(Table 7: 13% reduction in AUC, p=0.0156). For the rBPI₂₃ treatmentperiod, CI was 2.51±0.16 (L/min/m²) at time 0 and gradually rose to3.27±0.30 at 3:30 hours. For the placebo treatment period: CI was2.64±0.18 at time 0 rising to 3.98±0.27 at 3:30 hours. At 4:30 hours,cardiac indices were elevated in both study periods compared to baselineand both gradually returned to baseline leves by 12 hours.

Systolic (SBP), Diastolic (DBP), and mean arterial blood pressure (MAP)decreased in both study periods and were not influenced by the rBPI₂₃infusion.

Systemic vascular resistance index (SVRI) showed a decrease in bothperiods but the decrease was significantly less in the rBPI₂₃ infusionperiod (Table 7: 28% reduction in AUC, p=0.304). The SVRI was2714.4±204.0 at baseline falling to 1633.8±88.5 at 3 hours in theplacebo treatment period versus 2908.2±205.2 at baseline and2056.0±145.2 at 3 hours in the rBPI₂₃ treatment period (FIG. 18).

End diastolic volume and end systolic volume as measured by M-modeechocardiography were not different between treatment periods at anytimepoint. Average end diastolic volume showed a gradual decline from201±13 mL at baseline to 188±13 mL at 4 hours in the placebo period and188±11 mL at 4 hours in the rBPI₂₃ period. End systolic volume changedfrom 88±6 mL at baseline to 74±9 mL at 4 hours in the placebo period and75±7 mL at 4 hours in the rBPI₂₃ period.

M-mode ejection fraction showed a slight increase from 56±0.9% atbaseline to 60±2.8% at 4 hours and 59±1% at 12 hours in the rBPI₂₃treatment period versus 62±2.6% at 4 hours and 60±2.8% at 12 hours inthe placebo treatment period. Fractional shortening also showed anincrease from 33.1±0.7% at baseline to 36±2.3% at 4 hours and 35±0.9% at12 hours in the rBPI₂₃ treatment period versus 38±2.7% at 4 hours and37±2.2% at 12 hours in the placebo treatment period.

The above results establish that treatment of experimental endotoxemiain humans with a BPI protein product resulted in statisticallysignificant modification of hyperdynamic changes in left ventricularfunction in response to endotoxin in circulation. The BPI proteinproduct alleviated the decreases in systemic vascular resistance indexand the concomitant increase in cardiac index which attend presence ofendotoxin.

Numerous additional aspects and advantages of the present invention willbe apparent to those skilled in the art upon consideration of the aboveillustrative examples of presently preferred practice thereof. Forexample, it will be apparent that human patients suffering from Gramnegative bacteremia, accidental injection of endotoxin contaminatedfluids or systemic release of endotoxin by translocation from the gutwill benefit from administration of BPI protein products (by, e.g.,continuous intravenous infusion) to provide serologically andhematologically verifiable reduction in endotoxin mediated increases,for example, in levels of circulating cytokines and tissue plasminogenactivator and changes in numbers of lymphocytes. It will also beapparent that BPI protein product administration will provide abeneficial adjunctive therapy for patients being treated withantibiotics and encountering entry of endotoxin into circulation as aresult of bacterial lysis mediated by the antibiotic(s). Therefore onlysuch limitations as appear in the appended claims should be placed uponthe invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1813 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 31..1491                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 124..1491                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "rBPI"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CAGGCCTTGAGGTTTTGGCAGCTCTGGAGGATGAGAGAGAACATGGCCAGGGGC54                      MetArgGluAsnMetAlaArgGly                                                      31-30-25                                                                      CCTTGCAACGCGCCGAGATGGGTGTCCCTGATGGTGCTCGTCGCCATA102                           ProCysAsnAlaProArgTrpValSerLeuMetValLeuValAlaIle                              20-15- 10                                                                     GGCACCGCCGTGACAGCGGCCGTCAACCCTGGCGTCGTGGTCAGGATC150                           GlyThrAlaValThrAlaAlaValAsnProGlyValValValArgIle                              515                                                                           TCCCAGAAGGGCCTGGACTACGCCAGCCAGCAGGGGACGGCCGCTCTG198                           SerGlnLysGlyLeuAspTyrAlaSerGlnGlnGlyThrAlaAlaLeu                              10152025                                                                      CAGAAGGAGCTGAAGAGGATCAAGATTCCTGACTACTCAGACAGCTTT246                           GlnLysGluLeuLysArgIleLysIleProAspTyrSerAspSerPhe                              303540                                                                        AAGATCAAGCATCTTGGGAAGGGGCATTATAGCTTCTACAGCATGGAC294                           LysIleLysHisLeuGlyLysGlyHisTyrSerPheTyrSerMetAsp                              455055                                                                        ATCCGTGAATTCCAGCTTCCCAGTTCCCAGATAAGCATGGTGCCCAAT342                           IleArgGluPheGlnLeuProSerSerGlnIleSerMetValProAsn                              606570                                                                        GTGGGCCTTAAGTTCTCCATCAGCAACGCCAATATCAAGATCAGCGGG390                           ValGlyLeuLysPheSerIleSerAsnAlaAsnIleLysIleSerGly                              758085                                                                        AAATGGAAGGCACAAAAGAGATTCTTAAAAATGAGCGGCAATTTTGAC438                           LysTrpLysAlaGlnLysArgPheLeuLysMetSerGlyAsnPheAsp                              9095100105                                                                    CTGAGCATAGAAGGCATGTCCATTTCGGCTGATCTGAAGCTGGGCAGT486                           LeuSerIleGluGlyMetSerIleSerAlaAspLeuLysLeuGlySer                              110115120                                                                     AACCCCACGTCAGGCAAGCCCACCATCACCTGCTCCAGCTGCAGCAGC534                           AsnProThrSerGlyLysProThrIleThrCysSerSerCysSerSer                              125130135                                                                     CACATCAACAGTGTCCACGTGCACATCTCAAAGAGCAAAGTCGGGTGG582                           HisIleAsnSerValHisValHisIleSerLysSerLysValGlyTrp                              140145150                                                                     CTGATCCAACTCTTCCACAAAAAAATTGAGTCTGCGCTTCGAAACAAG630                           LeuIleGlnLeuPheHisLysLysIleGluSerAlaLeuArgAsnLys                              155160165                                                                     ATGAACAGCCAGGTCTGCGAGAAAGTGACCAATTCTGTATCCTCCAAG678                           MetAsnSerGlnValCysGluLysValThrAsnSerValSerSerLys                              170175180185                                                                  CTGCAACCTTATTTCCAGACTCTGCCAGTAATGACCAAAATAGATTCT726                           LeuGlnProTyrPheGlnThrLeuProValMetThrLysIleAspSer                              190195200                                                                     GTGGCTGGAATCAACTATGGTCTGGTGGCACCTCCAGCAACCACGGCT774                           ValAlaGlyIleAsnTyrGlyLeuValAlaProProAlaThrThrAla                              205210215                                                                     GAGACCCTGGATGTACAGATGAAGGGGGAGTTTTACAGTGAGAACCAC822                           GluThrLeuAspValGlnMetLysGlyGluPheTyrSerGluAsnHis                              220225230                                                                     CACAATCCACCTCCCTTTGCTCCACCAGTGATGGAGTTTCCCGCTGCC870                           HisAsnProProProPheAlaProProValMetGluPheProAlaAla                              235240245                                                                     CATGACCGCATGGTATACCTGGGCCTCTCAGACTACTTCTTCAACACA918                           HisAspArgMetValTyrLeuGlyLeuSerAspTyrPhePheAsnThr                              250255260265                                                                  GCCGGGCTTGTATACCAAGAGGCTGGGGTCTTGAAGATGACCCTTAGA966                           AlaGlyLeuValTyrGlnGluAlaGlyValLeuLysMetThrLeuArg                              270275280                                                                     GATGACATGATTCCAAAGGAGTCCAAATTTCGACTGACAACCAAGTTC1014                          AspAspMetIleProLysGluSerLysPheArgLeuThrThrLysPhe                              285290295                                                                     TTTGGAACCTTCCTACCTGAGGTGGCCAAGAAGTTTCCCAACATGAAG1062                          PheGlyThrPheLeuProGluValAlaLysLysPheProAsnMetLys                              300305310                                                                     ATACAGATCCATGTCTCAGCCTCCACCCCGCCACACCTGTCTGTGCAG1110                          IleGlnIleHisValSerAlaSerThrProProHisLeuSerValGln                              315320325                                                                     CCCACCGGCCTTACCTTCTACCCTGCCGTGGATGTCCAGGCCTTTGCC1158                          ProThrGlyLeuThrPheTyrProAlaValAspValGlnAlaPheAla                              330335340345                                                                  GTCCTCCCCAACTCCTCCCTGGCTTCCCTCTTCCTGATTGGCATGCAC1206                          ValLeuProAsnSerSerLeuAlaSerLeuPheLeuIleGlyMetHis                              350355360                                                                     ACAACTGGTTCCATGGAGGTCAGCGCCGAGTCCAACAGGCTTGTTGGA1254                          ThrThrGlySerMetGluValSerAlaGluSerAsnArgLeuValGly                              365370375                                                                     GAGCTCAAGCTGGATAGGCTGCTCCTGGAACTGAAGCACTCAAATATT1302                          GluLeuLysLeuAspArgLeuLeuLeuGluLeuLysHisSerAsnIle                              380385390                                                                     GGCCCCTTCCCGGTTGAATTGCTGCAGGATATCATGAACTACATTGTA1350                          GlyProPheProValGluLeuLeuGlnAspIleMetAsnTyrIleVal                              395400405                                                                     CCCATTCTTGTGCTGCCCAGGGTTAACGAGAAACTACAGAAAGGCTTC1398                          ProIleLeuValLeuProArgValAsnGluLysLeuGlnLysGlyPhe                              410415420425                                                                  CCTCTCCCGACGCCGGCCAGAGTCCAGCTCTACAACGTAGTGCTTCAG1446                          ProLeuProThrProAlaArgValGlnLeuTyrAsnValValLeuGln                              430435440                                                                     CCTCACCAGAACTTCCTGCTGTTCGGTGCAGACGTTGTCTATAAA1491                             ProHisGlnAsnPheLeuLeuPheGlyAlaAspValValTyrLys                                 445450455                                                                     TGAAGGCACCAGGGGTGCCGGGGGCTGTCAGCCGCACCTGTTCCTGATGGGCTGTGGGGC1551              ACCGGCTGCCTTTCCCCAGGGAATCCTCTCCAGATCTTAACCAAGAGCCCCTTGCAAACT1611              TCTTCGACTCAGATTCAGAAATGATCTAAACACGAGGAAACATTATTCATTGGAAAAGTG1671              CATGGTGTGTATTTTAGGGATTATGAGCTTCTTTCAAGGGCTAAGGCTGCAGAGATATTT1731              CCTCCAGGAATCGTGTTTCAATTGTAACCAAGAAATTTCCATTTGTGCTTCATGAAAAAA1791              AACTTCTGGTTTTTTTCATGTG1813                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 487 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetArgGluAsnMetAlaArgGlyProCysAsnAlaProArgTrpVal                              31-30-25-20                                                                   SerLeuMetValLeuValAlaIleGlyThrAlaValThrAlaAlaVal                              15-10-51                                                                      AsnProGlyValValValArgIleSerGlnLysGlyLeuAspTyrAla                              51015                                                                         SerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLys                              202530                                                                        IleProAspTyrSerAspSerPheLysIleLysHisLeuGlyLysGly                              354045                                                                        HisTyrSerPheTyrSerMetAspIleArgGluPheGlnLeuProSer                              50556065                                                                      SerGlnIleSerMetValProAsnValGlyLeuLysPheSerIleSer                              707580                                                                        AsnAlaAsnIleLysIleSerGlyLysTrpLysAlaGlnLysArgPhe                              859095                                                                        LeuLysMetSerGlyAsnPheAspLeuSerIleGluGlyMetSerIle                              100105110                                                                     SerAlaAspLeuLysLeuGlySerAsnProThrSerGlyLysProThr                              115120125                                                                     IleThrCysSerSerCysSerSerHisIleAsnSerValHisValHis                              130135140145                                                                  IleSerLysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys                              150155160                                                                     IleGluSerAlaLeuArgAsnLysMetAsnSerGlnValCysGluLys                              165170175                                                                     ValThrAsnSerValSerSerLysLeuGlnProTyrPheGlnThrLeu                              180185190                                                                     ProValMetThrLysIleAspSerValAlaGlyIleAsnTyrGlyLeu                              195200205                                                                     ValAlaProProAlaThrThrAlaGluThrLeuAspValGlnMetLys                              210215220225                                                                  GlyGluPheTyrSerGluAsnHisHisAsnProProProPheAlaPro                              230235240                                                                     ProValMetGluPheProAlaAlaHisAspArgMetValTyrLeuGly                              245250255                                                                     LeuSerAspTyrPhePheAsnThrAlaGlyLeuValTyrGlnGluAla                              260265270                                                                     GlyValLeuLysMetThrLeuArgAspAspMetIleProLysGluSer                              275280285                                                                     LysPheArgLeuThrThrLysPhePheGlyThrPheLeuProGluVal                              290295300305                                                                  AlaLysLysPheProAsnMetLysIleGlnIleHisValSerAlaSer                              310315320                                                                     ThrProProHisLeuSerValGlnProThrGlyLeuThrPheTyrPro                              325330335                                                                     AlaValAspValGlnAlaPheAlaValLeuProAsnSerSerLeuAla                              340345350                                                                     SerLeuPheLeuIleGlyMetHisThrThrGlySerMetGluValSer                              355360365                                                                     AlaGluSerAsnArgLeuValGlyGluLeuLysLeuAspArgLeuLeu                              370375380385                                                                  LeuGluLeuLysHisSerAsnIleGlyProPheProValGluLeuLeu                              390395400                                                                     GlnAspIleMetAsnTyrIleValProIleLeuValLeuProArgVal                              405410415                                                                     AsnGluLysLeuGlnLysGlyPheProLeuProThrProAlaArgVal                              420425430                                                                     GlnLeuTyrAsnValValLeuGlnProHisGlnAsnPheLeuLeuPhe                              435440445                                                                     GlyAlaAspValValTyrLys                                                         450455                                                                        __________________________________________________________________________

What is claimed is:
 1. A method for treatment of humans exposed tobacterial endotoxin in circulation comprising administering aBactericidal/Permeability Increasing Product (BPI) protein product in anamount effective to alleviate endotoxin mediated increase in circulatingtumor necrosis factor and interleukin
 6. 2. A method for treatment ofhumans exposed to bacterial endotoxin in circulation comprisingadministering a BPI protein product in an amount effective to alleviateendotoxin mediated increase in circulating interleukin 8 and inneutrophil degranulation as characterized by increased circulatinglactoferrin and/or elastase/α1 antitrypsin complexes.
 3. A method fortreatment of humans exposed to bacterial endotoxin in circulationcomprising administering a BPI protein product in an amount effective toalleviate endotoxin mediated changes in numbers of circulatinglymphocytes.
 4. A method for treatment of humans exposed to bacterialendotoxin in circulation comprising administering a BPI protein productin an amount effective to alleviate endotoxin mediated increase incirculating tissue plasminogen activator and tissue plasminogenactivator activity.
 5. A method for treatment of humans exposed tobacterial endotoxin in circulation comprising administering a BPIprotein product in an amount effective to alleviate endotoxin-mediateddecreases in systemic vascular resistance index.
 6. The method of claim1, 2, 3, 4, or 5 wherein the BPI protein product is administered in adosage amount of about 0.1 to 10 mg/kg of body weight.